Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.

The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Lu pour vous: Prévention cardiovasculaire: efficacité des statines chez les plus de 70 ans

Résumé de: Glynn RJ et al. Rosuvastatin for primary prevention in older persons with elevated C-reactive protein and low to average low-density lipoprotein cholesterol levels: exploratory analysis of a randomized trial. Ann Intern Med. 2010 Apr 20;152(8):488-96, PMID: 20404379.

Potentiometric titration as a method of determining the stoichiometric dissociation constants of weak acids from the thermodynamic dissociation constants of aqueous solutions at 298.15 K

The simple single-ion activity coefficient equation originating from the Debye-Hückel theory was used to determine the thermodynamic and stoichiometric dissociation constants of weak acids from data concerning galvanic cells. Electromotive force data from galvanic cells without liquid junctions, which was obtained from literature, was studied in conjuction with the potentiometric titration data relating to aqueous solutions at 298.15 K. The dissociation constants of weak acids could be determined by the presented techniques and almost all the experimental data studied could be interpreted within the range of experimental error. Potentiometric titration has been used here and the calculation methods were developed to obtain the thermodynamic and stoichiometric dissociation constants of some weak acids in aqueous solutions at 298.15 K. The ionic strength of titrated solutions were adjusted using an inert electrolyte, namely, sodium or potassium chloride. Salt content alonedetermines the ionic strength. The ionic strength of the solutions studied varied from 0.059 mol kg-1 to 0.37 mol kg-1, and in some cases up to 1.0 mol kg-1. The following substances were investigated using potentiometric titration: aceticacid, propionic acid, L-aspartic acid, L-glutamic acid and bis(2,2-dimethyl-3-oxopropanol) amine.

The AMPK family member Snf1 protects Saccharomyces cerevisiae cells upon glutatione oxidation

The AMPK/Snf1 kinase has a central role in carbon metabolism homeostasis in Saccharomyces cerevisiae. In this study, we show that Snf1 activity, which requires phosphorylation of the Thr210 residue, is needed for protection against selenite toxicity. Such protection involves the Elm1 kinase, which acts upstream of Snf1 to activate it. Basal Snf1 activity is sufficient for the defense against selenite, although Snf1 Thr210 phosphorylation levels become increased at advanced treatment times, probably by inhibition of the Snf1 dephosphorylation function of the Reg1 phosphatase. Contrary to glucose deprivation, Snf1 remains cytosolic during selenite treatment, and the protective function of the kinase does not require its known nuclear effectors. Upon selenite treatment, a null snf1 mutant displays higher levels of oxidized versus reduced glutathione compared to wild type cells, and its hypersensitivity to the agent is rescued by overexpression of the glutathione reductase gene GLR1. In the presence of agents such as diethyl maleate or diamide, which cause alterations in glutathione redox homeostasis by increasing the levels of oxidized glutathione, yeast cells also require Snf1 in an Elm1-dependent manner for growth. These observations demonstrate a role of Snf1 to protect yeast cells in situations where glutathione-dependent redox homeostasis is altered to a more oxidant intracellular environment and associates AMPK to responses against oxidative stress.

Data triangulation in the context of opioids monitoring via wastewater analyses.

BACKGROUND: The need to contextualise wastewater-based figures about illicit drug consumption by comparing them with other indicators has been stressed by numerous studies. The objective of the present study was to further investigate the possibility of combining wastewater data to conventional statistics to assess the reliability of the former method and obtain a more balanced picture of illicit drug consumption in the investigated area. METHODS: Wastewater samples were collected between October 2013 and July 2014 in the metropolitan area of Lausanne (226,000 inhabitants), Switzerland. Methadone, its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), the exclusive metabolite of heroin, 6-monoacetylmorphine (6-MAM), and morphine loads were used to estimate the amounts of methadone and heroin consumed. RESULTS: Methadone consumption estimated from EDDP was in agreement with the expectations. Heroin estimates based on 6-MAM loads were inconsistent. Estimates obtained from morphine loads, combined to prescription/sales data, were in agreement with figures derived from syringe distribution data and general population surveys. CONCLUSIONS: The results obtained for methadone allowed assessing the reliability of the selected sampling strategy, supporting its ability to capture the consumption of a small cohort (i.e., 743 patients). Using morphine as marker, in combination with prescription/sales data, estimates in accordance with other indicators about heroin use were obtained. Combining different sources of data allowed strengthening the results and suggested that the different indicators (i.e., administration route, average dosage and number of consumers) contribute to depict a realistic representation of the phenomenon in the investigated area. Heroin consumption was estimated to approximately 13gday(-1) (118gday(-1) at street level).

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

CNDOL: A fast and reliable method for the calculation of electronic properties of very large systems. Applications to retinal binding pocket in rhodopsin and gas phase porphine.

Very large molecular systems can be calculated with the so called CNDOL approximate Hamiltonians that have been developed by avoiding oversimplifications and only using a priori parameters and formulas from the simpler NDO methods. A new diagonal monoelectronic term named CNDOL/21 shows great consistency and easier SCF convergence when used together with an appropriate function for charge repulsion energies that is derived from traditional formulas. It is possible to obtain a priori molecular orbitals and electron excitation properties after the configuration interaction of single excited determinants with reliability, maintaining interpretative possibilities even being a simplified Hamiltonian. Tests with some unequivocal gas phase maxima of simple molecules (benzene, furfural, acetaldehyde, hexyl alcohol, methyl amine, 2,5 dimethyl 2,4 hexadiene, and ethyl sulfide) ratify the general quality of this approach in comparison with other methods. The calculation of large systems as porphine in gas phase and a model of the complete retinal binding pocket in rhodopsin with 622 basis functions on 280 atoms at the quantum mechanical level show reliability leading to a resulting first allowed transition in 483 nm, very similar to the known experimental value of 500 nm of "dark state." In this very important case, our model gives a central role in this excitation to a charge transfer from the neighboring Glu(-) counterion to the retinaldehyde polyene chain. Tests with gas phase maxima of some important molecules corroborate the reliability of CNDOL/2 Hamiltonians.

Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK.

The role of bacterial Hsp40, DnaJ, is to co-chaperone the binding of misfolded or alternatively folded proteins to bacterial Hsp70, DnaK, which is an ATP-fuelled unfolding chaperone. In addition to its DnaK targeting activity, DnaJ has a weak thiol-reductase activity. In between the substrate-binding domain and the J-domain anchor to DnaK, DnaJ has a unique domain with four conserved CXXC motives that bind two Zn(2+) and partly contribute to polypeptide binding. Here, we deleted in DnaJ this Zn-binding domain, which is characteristic to type I but not of type II or III J-proteins. This caused a loss of the thiol-reductase activity and strongly reduced the ability of DnaJ to mediate the ATP- and DnaK-dependent unfolding/refolding of mildly oxidized misfolded polypeptides, an inhibition that was alleviated in the presence of thioredoxin or DTT. We suggest that in addition to their general ability to target misfolded polypeptide substrates to the Hsp70/Hsp110 chaperone machinery, Type I J-proteins carry an ancillary protein dithiol-isomerase function that can synergize the unfolding action of the chaperone, in the particular case of substrates that are further stabilized by non-native disulfide bonds. Whereas the unfoldase can remain ineffective without the transient untying of disulfide bonds by the foldase, the foldase can remain ineffective without the transient ATP-fuelled unfolding of wrong local structures by the unfoldase.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children : Tracing the complexity of human postnatal steroidogenesis.

CONTEXT: Complex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by (a) the metabolites of the fetal-placental unit at birth, (b) the fetal adrenal androgens until its involution 3-6 months postnatally, and (c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. OBJECTIVE: The aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. METHODS: We conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. RESULTS: 61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. CONCLUSION: The urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.

Bartonella apis sp. nov., a honey bee gut symbiont of the class Alphaproteobacteria.

Here, we report the culture and characterization of an alphaproteobacterium of the order Rhizobiales, isolated from the gut of the honey bee Apis mellifera. Strain PEB0122T shares >95 % 16S rRNA gene sequence similarity with species of the genus Bartonella, a group of mammalian pathogens transmitted by bloodsucking arthropods. Phylogenetic analyses showed that PEB0122T and related strains from the honey bee gut form a sister clade of the genus Bartonella. Optimal growth of strain PEB0122T was obtained on solid media supplemented with defibrinated sheep blood under microaerophilic conditions at 35-37 °C, which is consistent with the cultural characteristics of other species of the genus Bartonella. Reduced growth of strain PEB0122T also occurred under aerobic conditions. The rod-shaped cells of strain PEB0122T had a mean length of 1.2-1.8 μm and revealed hairy surface structures. Strain PEB0122T was positive for catalase, cytochrome c oxidase, urease and nitrate reductase. The fatty acid composition was comparable to those of other species of the genus Bartonella, with palmitic acid (C16 : 0) and isomers of 18- and 19-carbon chains being the most abundant. The genomic DNA G+C content of PEB0122T was determined to be about 45.5 mol%. The high 16S rRNA gene sequence similarity with species of Bartonella and its close phylogenetic position suggest that strain PEB0122T represents a novel species within the genus Bartonella, for which we propose the name Bartonella apis sp. nov. The type strain is PEB0122T ( = NCIMB 14961T = DSM 29779T).

Electrocatalytic oxidation of beta-dicarbonyl compounds using ceric methanesulphonate as mediator

beta-dicarbonyl compounds were oxidized electrocatalytically, with fragmentation and loss of "ch2", using ceric methanesulphonate as a mediator. 2,4-pentanedione yields acetic acid (90%), methyl acetoacetate yields acetic acid (84%) plus methanol and dimethyl malonate yields methanol (64%). For 1,3-diphenyl-1,3-propanedione and 1,3-cyclohexanedione, benzoic acid (61% yield) and glutaric acid (75% yield) were obtained, respectively. Methyl cyanoacetate and malononitrile were inert.

Optical characterization of sol-gel glasses derived from Eu3+ complex-forming precursors

Fabrication of new optical devices based upon the incorporation of rare earth ions via sol-gel methods depends on elimination of dopant ion clusters and residual hydroxyl groups from the final material. The optical absorption and/or luminescence properties of luminescent rare earth ions are influenced by the local bonding environment and the distribution of the rare-earth dopants in the matrix. Typically, dopants are incorporated into gel via dissolution of soluble species into the initial precursor sol. In this work, Eu3+ is used as optical probe, to assess changes in the local environment. Results of emission, excitation, fluorescence line narrowing and lifetimes studies of Eu3+-doped gels derived from Si(OCH3)4 and fluorinated/chelate Eu3+ precursors are presented. The precursors used in the sol-gel synthesis were: tris (6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionate) Eu(III), Eu (III) trifluoromethanesulfonate, Eu(III) acetylacetonate hydrate, Eu (III) trifluoroacetate trihidrate, tris (2,2,6,6-tetramethyl-3,5- heptanedionate) Eu(III) and Eu(NO3)3.6H2O. The results were interpreted in terms of the evolution of the Eu3+ fluorescence in systems varying from solution to the gels densified to 800ºC. The lifetimes studies indicate that the fluorinated precursors are effective at reducing the water content in densified gels.